Hemoglobin solutions have potential application in injured man as oxygen-carrying plasma expanders in those situations in which the filling of the vascular system and the oxygen-transporting capacity of the blood are insufficient. The advantage over erythrocytes or blood is that the solutions are universally applicable without typing blood groups. A further advantage is that the hemoglobin solutions have a longer storage life. For this purpose, however, it is necessary to prepare and modify hemoglobin from erythrocytes in such a manner that the three main problems of hemoglobin solutions are minimized. These three problems are:
1) the presence of residues of membrane fragments (stroma) originating from erythrocytes, PA1 2) the high intrinsic oxygen affinity outside the environment of the erythrocyte, and PA1 3) the short retention time in the circulation because (dissociated) hemoglobin disappears rapidly from the vascular system because of leakage through the kidneys. PA1 1) the half-life period in the circulation of man will be about 8 hours, whereas 24 hours is desirable, and PA1 2) the oxygen-transporting capacity cannot be more than half of that of normal blood, because the free hemoglobin can only be present in a concentration of maximally 6-7 g/100 ml in the circulation in view of the colloid-osmotic pressure.
As early as 1967, Rabiner described a technique for the preparation of hemoglobin solutions in which stroma was removed by filtration and centrifugation so that earlier observed coagulation problems encountered in the kidneys of dogs could be avoided (J. Expl. Med. 126, 1127-1142 (1967). This method for the removal of stroma was subsequently found to be inadequate to obviate many of the side effect encountered.
In 1972 Benesch et al, (Biochemistry 11, 3576-3582 (1972)) determined that a marked decrease of oxygen affinity could be realized by covalently binding pyridoxal 5-phosphate (PLP) to hemoglobin. This permanent bonding was found to prevent PLP from leaving the free hemoglobin infused into the circulation, as would be the case with 2,3-di-phosphoglycerate (2,3-DPG). PLP can also be bound at different sites to one of the .beta.-globin chains in the 2,3-DPG binding cavity.
By polymerizing hemoglobin, the retention time in the circulation can be prolonged. This is described by Mazur et al (U.S. Pat. No. 3,925,344 (1975)) with (m) ethyl glutarimidate, (m) ethyl succinimidate and (m) ethyl suberimidate, etc. and by Bonsen et al (U.S. Pat. Nos. 4,001,200, 4,001,401, 4,053,590 and 4,061,736 (1977)), inter alia with different dialdehydes, particularly glutaraldehyde- A problem encountered with these modifications of hemoglobin is that the oxygen affinity substantially increases so that the oxygen release in vivo to the tissues cannot be optimal. By both coupling hemoglobin to PLP and polymerizing hemoglobin with glutaraldehyde Bonhard et al (U.S. Pat. Nos. 4,136,093 and 4,336,248 (1979)) have obtained a slight improvement in the oxygen affinity in combination with a prolonged retention time. Rausch et al (WO 88/03408) describe polymerization of bovine hemoglobin in which a long circulation time in test animals has been shown. The difference in species, however, will stand in the way of administration to human beings.
Notwithstanding these improvements, clinical application had not yet been relied upon since the administration of small amounts of hemoglobin solution to healthy volunteers showed toxic effects (Savitsky et al, Clin. Pharmacol. Ther. 23, 73-90 (1978)). More recently, however, a group led by Moss administered a comparable dose to healthy volunteers with no side effects being observed. The hemoglobin solution then used was a polymerization product of HbPLP analogous to that produced according to Bonhard but improved as described in WO 87/07832 (Sehgal et al). According to this patent, unconverted hemoglobin must be removed from the polymerization mixture (to prevent negative effects on the renal function), which is achieved by ultrafiltration, gel filtration, and affinity chromatography.
Still another approach to prolonging the retention time in the circulation and decreasing the oxygen affinity is intramolecular cross-linking. Thus, dissociation of tetrameric Hb into dimers and leakage through the kidneys are prevented. Cross-linking of the .beta.-chains was first shown to prolong the retention time by Bunn et al with the use of bis(N-maleidomethyl)ether (J. Exp. Med. 129, 909-934 (1969)). This modification, however, led to a considerable increase in the oxygen affinity. Bakker et al (Adv. Exp. Med. Biol. 180, 345-356 (1985)) showed that by coupling with 2-nor-2-formylpyridoxal 5-phosphate (NFPLP; see formula 1 of the sheet of formulae), as described by Benesch et al (Biochem. Biophys. Res. Commun. 63, 1123-1129 (1975)), both a longer retention time and a lower oxygen affinity are realized. Walder et al use diaspirin compounds for coupling .alpha.-globin chains (US patents 4,598,604 and 4,600,531 (1986)). The oxygen affinity of their coupling product is equal to that of erythrocytes and the retention time in the circulation is prolonged by a factor of 3. Bucci et al (U.S. Pat. No. 4,584,130 (1986)) couple hemoglobin intramolecularly to negatively charged organic reagents. Kavanaugh et al use the bifunctional reagent 4,4'-diisothiocyanostibene-2,2'-disulfonate (Biochemistry 27, 1804-1808 (1988)). Their coupling product has a physiologic oxygen affinity but is obtained in low yield.
The modification closest to the physiologic situation is the coupling with NFPLP or later mentioned analogous compounds. This led to coupling of the .beta.-chains through valine-1 of the first .beta.-chain and lysine-87 of the second .beta.-chain (Arnone et al, J. Mol. Biol. 115, 627-642 (1977)). The NFPLP is covalently bound and is permanently present at the site where 2,3-DPG in the erythrocytes influences the oxygen affinity. This .beta.-chain coupling was first described by Benesch et al in 1975 and elaborated further by Van der Plas et al (J. Lab. Clin. Med. 108, 253-360 (1986, Transfusion 27, 425-430 (1987), Transfusion. 28, 525-530 (1988)). They found a substantially decreased oxygen affinity, which guarantees a proper oxygen release under the most frequent conditions. The retention time in the circulation is prolonged by a factor of 3 (Bleeker et al, J. Lab. Clin. Med. 108, 448-455 (1986)). As a plasma expander with oxygen-transporting capacity, however, the cross-linked hemoglobin (HbNFPLP) is open to criticism for the following reasons: